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Pathway Activation (pathway + activation)
Selected AbstractsEmx3 is required for the differentiation of dorsal telencephalic neuronsDEVELOPMENTAL DYNAMICS, Issue 8 2009Gudrun Viktorin Abstract emx3 is first expressed in prospective telencephalic cells at the anterior border of the zebrafish neural plate. Knockdown of Emx3 function by morpholino reduces the expression of markers specific to dorsal telencephalon, and impairs axon tract formation. Rescue of both early and late markers requires low-level expression of emx3 at the one- or two-somite stage. Higher emx3 expression levels cause dorsal telencephalic markers to expand ventrally, which points to a possible role of emx3 in specifying dorsal telencephalon and a potential new function for Wnt/beta-catenin pathway activation. In contrast to mice, where Emx2 plays a major role in dorsal telencephalic development, knockdown of zebrafish Emx2 apparently does not affect telencephalic development. Similarly, Emx1 knockdown has little effect. Previously, emx3 was thought to be fish-specific. However, we found all three emx orthologs in Xenopus tropicalis and opossum (Monodelphis domestica) genomes, indicating that emx3 was present in an ancestral tetrapod genome. Developmental Dynamics 238:1984,1998, 2009. © 2009 Wiley-Liss, Inc. [source] Advanced glycation end products-induced apoptosis attenuated by PPAR, activation and epigallocatechin gallate through NF-,B pathway in human embryonic kidney cells and human mesangial cellsDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2010Yao-Jen Liang Abstract Background Diabetic nephropathy has attracted many researchers' attention. Because of the emerging evidence about the effects of advanced glycation end products (AGEs) and receptor of AGE (RAGE) on the progression of diabetic nephropathy, a number of different therapies to inhibit AGE or RAGE are under investigation. The purpose of the present study was to examine whether peroxisome proliferator-activated receptor , (PPAR,) agonist (L-165041) or epigallocatechin gallate (EGCG) alters AGE-induced pro-inflammatory gene expression and apoptosis in human embryonic kidney cells (HEK293) and human mesangial cells (HMCs). Methods The HEK cells and HMC were separated into the following groups: 100 µg/mL AGE alone for 18 h; AGE treated with 1 µM L-165041 or 10 µM EGCG, and untreated cells. Inflammatory cytokines, nuclear factor-,B pathway, RAGE expression, superoxide dismutase and cell apoptosis were determined. Results AGE significantly increased tumour necrosis factor-, (TNF-,), a major pro-inflammatory cytokine. The mRNA and protein expression of RAGE were up-regulated. These effects were significantly attenuated by pre-treatment with L-165041 or EGCG. AGE-induced nuclear factor-,B pathway activation and both cells apoptosis were also inhibited by L-165041 or EGCG. Furthermore, both L-165041 and EGCG increased superoxide dismutase levels in AGE-treated HEK cells and HMC. Conclusions This study demonstrated that PPAR, agonist and EGCG decreased the AGE-induced kidney cell inflammation and apoptosis. This study provides important insights into the molecular mechanisms of EGCG and PPAR, agonist in attenuation of kidney cell inflammation and may serve as a therapeutic modality to treat patients with diabetic nephropathy. Copyright © 2010 John Wiley & Sons, Ltd. [source] Role of the complement-lectin pathway in anaphylactoid reaction induced with lipopolysaccharide in miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2003wierzko Abstract We show that Proteus vulgaris,O25 (PO25) lipopolysaccharide (LPS) induced an anaphylactoid reaction not only in wild-type and in lipid,A non-responding mice but also in recombinase-activating gene-2-deficient (RAG-2,/,) and in mast cell-deficient (W/Wv) animals. Western blot analysis indicated that PO25 LPS bound to Ra-reactive factor (RaRF), the complex of mannan-binding lectins (MBL) and MBL-associated serine proteases. Binding of RaRF to PO25 LPS led to the activation of C4 component without participation of either C1 or Ig, via the lectin pathway. Relative concentration of RaRF and hemolytic activity in mouse serum decreased rapidly during the process of anaphylactoid reaction. A significant drop of MBL-A, but not MBL-C level was observed. Administrationwith antiserum to RaRF prevented animals from death as a consequence of the inhibition of interaction of RaRF with the carbohydrate target and complement activation. These results indicate that complement-lectin pathway activation is responsible for the anaphylactoid reaction induced with LPS in muramyldipeptide-primed mice. RaRF also activated fibrinogen in vitro suggesting the involvement of the coagulation system in the process investigated. [source] Progestin upregulates G-protein-coupled receptor 30 in breast cancer cellsFEBS JOURNAL, Issue 10 2002Tytti M. Ahola A differential display method was used to study genes the expression of which is altered during growth inhibition induced by medroxyprogesterone acetate (MPA). A transcript of G-protein-coupled receptor 30 (GPR30) was upregulated by MPA in estrogen-treated MCF-7 breast cancer cells. Northern-blot analysis showed a progestin-specific primary target gene, which was enhanced by progesterone and different progestins, but not by dihydrotestosterone or dexamethasone, and which was abrogated by antiprogestin RU486. The dose-dependent and time-dependent increase in GPR30 mRNA expression correlated with MPA-induced growth inhibition in MCF-7 cells. Additionally, GPR30 upregulation by progestin correlated with growth inhibition when a comparison was made between different breast cancer cell lines. The ERK1/ERK2 pathway is capable of inducing progesterone receptor-dependent and ligand-dependent transcription. Thus we sought to establish whether different MAPK pathway inhibitors affect progestin-induced GPR30 mRNA regulation. The regulation of GPR30 was independent of ERK pathway activation, but the p38 pathway inhibitor induced GPR30 expression, which suggested a potential gene regulation pathway. These data demonstrate a new progestin target gene, the expression of which correlates with growth inhibition. [source] Complement Activation in Emergency Department Patients With Severe SepsisACADEMIC EMERGENCY MEDICINE, Issue 4 2010John G. Younger Abstract Objectives:, This study assessed the extent and mechanism of complement activation in community-acquired sepsis at presentation to the emergency department (ED) and following 24 hours of quantitative resuscitation. Methods:, A prospective pilot study of patients with severe sepsis and healthy controls was conducted among individuals presenting to a tertiary care ED. Resuscitation, including antibiotics and therapies to normalize central venous and mean arterial pressure (MAP) and central venous oxygenation, was performed on all patients. Serum levels of Factor Bb (alternative pathway), C4d (classical and mannose-binding lectin [MBL] pathway), C3, C3a, and C5a were determined at presentation and 24 hours later among patients. Results:, Twenty patients and 10 healthy volunteer controls were enrolled. Compared to volunteers, all proteins measured were abnormally higher among septic patients (C4d 3.5-fold; Factor Bb 6.1-fold; C3 0.8-fold; C3a 11.6-fold; C5a 1.8-fold). Elevations in C5a were most strongly correlated with alternative pathway activation. Surprisingly, a slight but significant inverse relationship between illness severity (by sequential organ failure assessment [SOFA] score) and C5a levels at presentation was noted. Twenty-four hours of structured resuscitation did not, on average, affect any of the mediators studied. Conclusions:, Patients with community-acquired sepsis have extensive complement activation, particularly of the alternative pathway, at the time of presentation that was not significantly reversed by 24 hours of aggressive resuscitation. ACADEMIC EMERGENCY MEDICINE,2010; 17:353,359 © 2010 by the Society for Academic Emergency Medicine [source] TGF-,1/SMAD signaling induces astrocyte fate commitment in vitro: Implications for radial glia developmentGLIA, Issue 10 2007Joice Stipursky Abstract Radial glial (RG) cells are specialized type of cell, which functions as neuronal precursors and scaffolding guides to migrating neurons during cerebral cortex development. After neurogenesis and migration are completed, most of RG cells transform into astrocytes. Mechanism and molecules involved in this process are not completely elucidated. We previously demonstrated that neurons activate the promoter of the astrocyte maturation marker GFAP in astrocytes by secretion of transforming growth factor beta 1 (TGF-,1) in vitro. Here, we studied the role of neurons and TGF-,1 pathway in RG differentiation. To address this question, we employed cortical progenitor cultures enriched in GLAST/nestin double-labeled cells, markers of RG cells. TGF-,1 and conditioned medium derived from neuron-astrocyte cocultures (CM) decreased the number of cells expressing the precursor marker nestin and increased that expressing GFAP in cortical progenitor cultures. These events were impaired by addition of neutralizing antibodies against TGF-,1. Increase in the number of GFAP positive cells was associated with Smads 2/3 nuclear translocation, a hallmark of TGF-,1 pathway activation. PCR-assays revealed a decrease in the levels of mRNA for the RG marker, BLBP (brain lipid binding protein), due to TGF-,1 and CM treatment. We further identified TGF-,1 receptor in cortical progenitor cultures suggesting that these cells might be target for TGF-,1 during development. Our work provides strong evidence that TGF-,1 might be a novel factor involved in RG-astrocyte transformation and highlights the role of neuron-glia interaction in this process. © 2007 Wiley-Liss, Inc. [source] Subtoxic N -methyl- D -aspartate delayed neuronal death in ischemic brain injury through TrkB receptor- and calmodulin-mediated PI-3K/Akt pathway activationHIPPOCAMPUS, Issue 7 2007Jing Xu Abstract Previous studies have shown that subtoxic NMDA moderated the neuronal survival in vitro and vivo. We performed this experiment to clarify the precise mechanism underlie subtoxic NMDA delayed neuronal death in ischemic brain injury. We found that pretreatment of NMDA (100 mg/kg) increased the number of the surviving CA1 pyramidal cells of hippocampus at 5 days of reperfusion. This dose of NMDA could also enhance Akt activation after ischemia/reperfusion (I/R). Here, we examined the possible mechanism that NMDA induced Akt activation. On the one hand, we found NMDA receptor-mediated Akt activation was associated with increased expression of BDNF (brain-derived neurotrophic factor) and activation of its high-affinity receptor TrkB after I/R in the hippocampus CA1 region, which could be held down by TrkB receptor antagonist K252a. On the other hand, we found that NMDA enhanced the binding of Ca2+ -dependent calmodulin (CaM) to p85 (the regulation subunit of PI-3K), which led to the activation of Akt. W-13, an active CaM inhibitor, prevented the combination of CaM and p85 and subsequent Akt activation. Furthermore, NMDA receptor-mediated Akt activation was reversed by combined treatment with LY294002, the specific blockade of PI-3K. Taken together, our results suggested that subtoxic NMDA exerts the neuroprotective effect via activation of prosurvival PI-3K/Akt pathway against ischemic brain injury, and BDNF-TrkB signaling and Ca2+ -dependent CaM cascade might contribute to NMDA induced activation of PI-3K/Akt pathway. © 2007 Wiley-Liss, Inc. [source] Defining a role for Sonic hedgehog pathway activation in desmoplastic medulloblastoma by identifying GLI1 target genesINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Joon Won Yoon Abstract A subgroup of medulloblastomas shows constitutive activation of the Sonic hedgehog pathway with expression of GLI1. We identified the subset of GLI1 transforming target genes specifically expressed in medulloblastomas by comparing GLI1 targets in RK3E cells transformed by GLI1 with the gene expression profile of Sonic hedgehog signature medulloblastomas. We identified 1,823 genes whose expression was altered more than 2-fold in 2 independent RK3E + GLI1 cell lines. We identified 25 whose expression was altered similarly in medulloblastomas expressing GLI1. We identified potential GLI binding elements in the regulatory regions of 10 of these genes, confirmed that GLI1 binds the regulatory regions and activates transcription of select genes, and showed that GLI1 directly represses transcription of Krox-20. We identified upregulation of CXCR4, a chemokine receptor that plays roles in the proliferation and migration of granule cell neuron precursors during development, supporting the concept that reinitiation of developmental programs may contribute to medulloblastoma tumorigenesis. In addition, the targets suggest a pathway through which GLI1 may ultimately affect medulloblastoma cell proliferation, survival and genomic stability by converging on p53, SGK1, MGMT and NTRK2. We identify a p53 mutation in RK3E + GLI1 cells, suggesting that p53 mutations may sometimes shift the balance toward dysregulated tumor cell survival. © 2008 Wiley-Liss, Inc. [source] ORIGINAL ARTICLE: The approach to the mechanism of calcitonin gene-related peptide-inducing inhibition of food intakeJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 5 2010J.-Y. Sun Summary The aim of this study was to investigate the anorectic mechanism of calcitonin gene-related peptide (CGRP) in rats. Intraperitoneal injection of CGRP (50 ,g/kg) resulted in decline (p < 0.05) in the food intake of rats at 0.5, 1, 2 and 4 h in comparison with saline control. Compared with saline-treated group, the levels of hypothalamic 3,,5,-cyclic adenosine monophosphate (cAMP) and plasma glucagon were increased (p < 0.05) in CGRP-treated group, but insulin level was decreased (p < 0.05). No significant changes (p > 0.05) in the plasma leptin were observed between two treatment groups. Calcitonin gene-related peptide injection down regulated (p < 0.05) both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) genes at mRNA levels, but up regulated (p < 0.05) the expression of cholecystokinin (CCK) gene. The correlations analysis showed that food intake was negatively correlated (p < 0.05) with CCK mRNA, cAMP and glucagon levels. Moreover, there existed negative correlations (p < 0.05) between MCH mRNA and glucagon levels, and positive correlations (p < 0.05) between insulin and leptin levels. The results showed that cAMP acting as the second messenger may play a vital role in the anorectic effects of CGRP. Calcitonin gene-related peptide could stimulate anorexigenic neuropeptides (i.e. CCK) and/or inhibit orexigenic neuropeptides (i.e. NPY and MCH) expression, and ultimately suppressed food intake that was functionally coupled to cAMP/PKA pathway activation. [source] Angiotensin II/angiotensin II type I receptor (AT1R) signaling promotes MCF-7 breast cancer cells survival via PI3-kinase/Akt pathwayJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Yanbin Zhao Angiotensin II (Ang II) is a bioactive peptide of the renin,angiotensin system, acting not only as a vasoconstrictor but also as a growth promoter via angiotensin II type 1 receptor (AT1R) in some cancer. In this study, we examined the mechanisms by which Ang II affected the cell proliferation in AT1R-positive MCF-7 human breast cancer cells. Ang II stimulated the growth of breast cancer cells in a dose- and time-dependent manner. The maximal proliferation effect on MCF-7 cells was obtained with 10,4,M Ang II at 24,h. Losartan (10,5,M, an AT1R antagonist) significantly decreased the level of Ang-II-induced proliferative effects, whereas PD123319 (10,5,M, an AT2R antagonist) had no effects. Moreover, Ang II could significantly accelerate S-phase progression, which was inhibited by losartan (10,5,M) or LY294002 (50,µM, a PI3-kinase inhibitor). In addition, Ang II caused rapid activation of p-Akt in a dose- and time-dependent manner. 10,4,M Ang II induced a significant increase of p-Akt in 15,min. The peak level of p-Akt could be persisted for at least 6,h. Among the signaling molecules downstream of Akt, we revealed that Ang II also significantly upregulated CyclinD1, GSK3,, and downregulated p27. Pretreatment with losartan (10,5,M) or LY294002 (50,µM) could significantly suppress these effects of Ang II. These findings suggest that Ang II plays a role in the growth of AT1R-positive breast cancer cells through PI3-kinase/Akt pathway activation. Therefore, targeting Ang II/AT1R signaling could be a novel therapeutic for breast cancer. J. Cell. Physiol. 225: 168,173, 2010. © 2010 Wiley-Liss, Inc. [source] Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004S. Marchetti The p42/p44 mitogen activated protein kinase (MAPK) pathway participates in a wide range of cellular programs including proliferation, migration, differentiation, and survival. Specific pharmacological inhibitors, like PD98059 and U0126, are often used to inhibit p42/p44 MAPK signaling. However, these inhibitors are not appropriate to study the function of these kinases in whole organisms. We thus developed an inducible system designed to inhibit p42/p44 MAPK activity through the expression of a phosphatase specific for these two kinases, the MAPK phosphatase 3 (MKP-3). A fibroblast cell line was established in which MKP-3 expression is controlled by tetracycline. Tetracycline-induced MKP-3 resulted in partial de-phosphorylation of p42/p44 MAPKs in serum-stimulated cells. However, we could improve MKP-3 stability and thereby the rate of MAPK de-phosphorylation, when the C-terminal end of MKP-3 was fused to the green fluorescent protein (GFP). Importantly, the fusion of GFP to MKP-3 did not alter the specificity of the phosphatase towards its MAPK substrates. We further show that conditional expression of MKP-3-GFP in this fibroblast cell line results in the inhibition of: (a) the phosphorylation of the p42/p44 MAPK substrates Elk1 and HIF-1,, (b) vascular endothelial growth factor (VEGF), cyclin D1, and c-fos gene transcription in response to MAPK pathway activation, and (c) cell proliferation. Finally, the MKP-3-GFP inducible cell line was transformed by Ha-ras and injected into nude mice. Treatment of mice with the tetracycline analog doxycycline resulted in a large delay in tumor emergence and growth as compared to the untreated control group, indicating that MKP-3-GFP activity is maintained in vivo. Altogether, these results show that inducible expression of MKP-3-GFP constitutes a valuable tool to study the role of p42/p44 MAPKs in various cellular responses in both cultured cell and animal models, a tool that may also be used to block unwanted cell growth in pathological conditions. © 2004 Wiley-Liss, Inc. [source] Cold-induced Glutamate Release in vivo from the Magnocellular Region of the Paraventricular Nucleus is Involved in Ovarian Sympathetic ActivationJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2010P. Jara We previously reported that centrally-induced sympathetic activation in response to cold stress is associated with a polycystic ovarian condition in rats, and thyrotrophin-releasing hormone (TRH) released locally from the magnocellular region of the paraventricular nucleus (PVN) appears to be involved in this activation. Because TRH neurones express NMDA glutamate receptors, in the present study, we investigated the role of glutamate in the increased release of TRH from magnocellular neurones induced by cold stress and its relationship to ovarian neurotransmission. Animals with a push,pull cannula stereotaxically implanted into the magnocellular portion of the PVN were exposed to cold stress (4 °C for 64 h) and subjected to intracerebral perfusion. Perfusate fractions were obtained and analysed by high-performance liquid chromatography to measure glutamate and GABA levels. Glutamate, but not GABA, release increased significantly in animals perfused under cold exposure. In vivo administration of glutamate to the PVN increased TRH release. Injection of MK-801 into the magnocellular portion of the PVN reduced ovarian noradrenaline turnover and led to an increase in catecholamine concentration from the adrenal glands and celiac ganglia. Taken together, the results obtained in the present study strongly suggest that glutamate release from the magnocellular PVN is sensitive to cold stress and that glutamate acts through the NMDA receptor to mediate cold-induced TRH release. This in turn triggers hypothalamic-ovarian pathway activation, which might be responsible for the polycystic condition induced by cold stress and other ovarian pathologies characterised by increased sympathetic discharge. [source] Acute and Chronic Alcohol Exposure Impair the Phagocytosis of Apoptotic Cells and Enhance the Pulmonary Inflammatory ResponseALCOHOLISM, Issue 10 2010Darren M. Boé Background:, Alcohol abuse increases the risk for acute respiratory distress syndrome (ARDS). Efferocytosis, the clearance of apoptotic cells, is important in the resolution of inflammation and is regulated by RhoA and rho kinase (ROCK) activation. The effects of alcohol on pulmonary Rho pathway activation and efferocytosis have not been determined. We hypothesize that acute and chronic alcohol exposure impair pulmonary efferocytosis, leading to heightened inflammation during ARDS. Methods:, For in vivo experiments, C57BL/6 mice received either a single intraperitoneal injection of alcohol or chronic ethanol-in-water for 8 weeks prior to intratracheal instillation of apoptotic cells or lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed for cells counts, calculation of the phagocytic index (PI), and Rho activity measurements. For in vitro studies, primary alveolar macrophages were cultured in alcohol (25,100 mM) and then co-cultured with apoptotic cells. RhoA activity was determined following alcohol exposure, and the PI was determined before and after treatment with the ROCK inhibitor, Y27632. Results:, Acute alcohol exposure was associated with impaired efferocytosis. Following LPS exposure, acute alcohol exposure was also associated with increased BAL neutrophils. Chronic alcohol exposure alone did not alter efferocytosis. However, following exposure to LPS, chronic alcohol exposure was associated with both impaired efferocytosis and increased BAL neutrophils. In vitro alcohol exposure caused a dose-dependent decrease in efferocytosis. Despite the fact that RhoA activity was decreased by alcohol exposure and RhoA inhibition did not alter the effects of alcohol on efferocytosis, treatment with the Rho kinase inhibitor, Y27632, reversed the effects of alcohol on efferocytosis. Conclusions:, Acute alcohol exposure impairs pulmonary efferocytosis, whereas exposure to chronic alcohol is only associated with impaired efferocytosis following LPS-induced lung injury. Both forms of alcohol exposure are associated with increased alveolar neutrophil numbers in response to LPS. The acute effects of alcohol on efferocytosis appear to be mediated, at least in part, by RhoA-independent activation of ROCK. Further studies are needed to dissect the differences between the effects of acute and chronic alcohol exposure on efferocytosis and to determine the effects of alcohol on alternative activators of ROCK. [source] Ras-mediated intestinal epithelial cell transformation requires cyclooxygenase-2-induced prostaglandin E2 signalingMOLECULAR CARCINOGENESIS, Issue 12 2007Gretchen A. Repasky Abstract Ras-mediated transformation is associated with upregulation of cyclooxygenase-2 (COX-2), which in turn promotes prostaglandin E2 (PGE2) synthesis and secretion. Although recent studies have identified molecular mechanisms by which Ras mediates upregulation of COX-2, conflicting observations have been made. Furthermore, while COX-2 upregulation has been shown to be important for Ras transformation, the signaling pathways initiated by PGE2 -stimulation of EP family of heterotrimeric G protein-coupled receptors (GPCR) and contribution of PGE2 signaling to Ras-mediated transformation are issues that remain unresolved. In this study, we first determined that Raf effector pathway activation of the extracellular-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade alone was sufficient and necessary for COX-2 and PGE2 upregulation. However, Raf-independent regulation of the c- jun N-terminal kinase (JNK) and p38 MAPK cascades is also involved in COX-2 and PGE2 upregulation, with the JNK and p38 pathways exhibiting opposing roles in COX-2 and PGE2 upregulation. Furthermore, in contrast to previous studies, we found that an epidermal growth factor (EGF) receptor autocrine growth mechanism, another Raf-independent signaling mechanism, was necessary for COX-2 and PGE2 upregulation. Second, we determined that inhibition of EP1/2 receptor function blocked growth transformation by Ras, demonstrating that PGE2 upregulation is a key transforming function of COX-2. Finally, we found that PGE2 stimulated the activation of Ras and ERK, but not Akt, and reduced matrix deprivation-induced apoptosis, in untransformed epithelial cells. In summary, our studies define additional, multiple signaling mechanisms that promote COX-2 and PGE2 expression and show that COX-2-stimulated PGE2 -EP receptor signaling is required for growth and survival transformation by Ras. © 2007 Wiley-Liss, Inc. [source] Complement analysis in children with idiopathic membranoproliferative glomerulonephritis: A long-term follow-upPEDIATRIC ALLERGY AND IMMUNOLOGY, Issue 3 2001Rainer Schwertz Fifty children with idiopathic membranoproliferative glomerulonephritis (MPGN), aged 2,14 years at apparent onset, were monitored for the presence of C3 nephritic factor (C3 NeF) and signs of complement activation in serum. In addition, C3 allotyping was performed in 32 patients. Observation time ranged from 2 to 20 (median 11) years. C3 NeF activity was detected at least once in 60% of the patients (in 11 of 26 with type I, in 15 of 17 with type II, and in four of seven with type III). C3 NeF-positive patients had significantly reduced levels of CH50 and C3 and elevated levels of C3dg/C3d. During follow-up, C3 levels were persistently normal in 62% of the patients with MPGN type I and in 43% with type III but in only 18% with type II. C3 allotype frequencies differed from those found in healthy controls with a significant shift to the C3F/C3FS variants in C3 NeF-positive patients. C3b(Bb)P as a marker for alternative pathway activation was not increased in C3 NeF-positive patients. Despite the presence of C3 NeF activity, C3 levels remained normal in six patients throughout the observation period. C3 NeF became undetectable in six patients, whereas seven developed C3 NeF activity during follow-up. There was no significant difference in renal survival probability in patients with or without C3 NeF activity. Neither C3 variants nor continous low C3 or low CH50 levels had any prognostic value for the clinical outcome. No factor H deficiency was detected. [source] Requirement for Metalloproteinase-dependent ERK and AKT Activation in UVB-induced G1-S Cell Cycle Progression of Human KeratinocytesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009Weinong Han UVB (280,315 nm) in natural sunlight represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here we demonstrate that low doses of UVB induce keratinocyte proliferation and cell cycle progression of human HaCaT keratinocytes. Different from UVA, UVB irradiation induced extracellular signal-regulated kinase (ERK) and AKT activation and their activation are both required for UVB-induced cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVB exposure and is upstream of ERK/AKT/cyclin D1 pathway activation and cell cycle progression following UVB radiation. Furthermore, metalloproteinase (MP) inhibitor GM6001 blocked UVB-induced ERK and AKT activation, cell cycle progression, and decreased the EGFR phosphorylation, demonstrating that MPs mediate the EGFR/ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB radiation. In addition, ERK or AKT activation is essential for EGFR activation because ERK or AKT inhibitor blocks EGFR activation following UVB radiation, indicating that EGFR/AKT/ERK pathways form a regulatory loop and converge into cell cycle progression following UVB radiation. Identification of these signaling pathways in UVB-induced cell cycle progression of quiescent keratinocytes as a process mimicking tumor promotion in vivo will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer. [source] Positive crosstalk between ERK and p38 in melanoma stimulates migration and in vivo proliferationPIGMENT CELL & MELANOMA RESEARCH, Issue 1 2009Yeriel Estrada Summary Melanoma is one of the most therapy-resistant cancers. Activating mutations in BRAF and NRAS are the source of extracellular signal regulated protein kinase (ERK) pathway activation. We show that melanoma cell lines, originating in different metastatic sites, with BRAF or NRAS mutations, in addition to active mitogen activated protein kinase (MAPK),ERK, also have highly activated stress activated protein kinase (SAPK)-p38. This is in direct contrast to carcinoma cells in which the activity of the two kinases appears to be mutually exclusive; high level of p38 activity inhibits, through a negative feedback, ERK activity and prevents tumorigenesis. Melanomas are insensitive to ERK inhibition by p38 and utilize p38-signaling pathway for migration and growth in vivo. We found a positive functional loop linking the high ERK activity to surface expression of ,V,3-integrin. This integrin, by interacting with vitronectin, induces p38 activity and increases IL-8 production, enhancing cell migration. Because the negative loop from p38 to ERK is lost, the two kinases can remain simultaneously activated. Inhibition of ERK and p38 activities is more effective in blocking in vivo growth than inhibition of each kinase individually. Future therapies might have to consider targeting of both pathways. [source] Signal pathway profiling of prostate cancer using reverse phase protein arraysPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2003Robert L. Grubb Abstract Reverse phase protein arrays represent a new proteomics microarray technology with which to study the fluctuating state of the proteome in minute quantities of cells. The activation status of cell signaling pathways controls cellular fate and deregulation of these pathways underpins carcinogenesis. Changes in pathway activation that occur between early stage prostatic epithelial lesions, prostatic stroma and the extracellular matrix can be analyzed by obtaining pure populations of cell types by laser capture microdissection (LCM) and analyzing the relative states of several key phosphorylation points within the cellular circuitry. We have applied reverse phase protein array technology to analyze the status of key points in cell signaling involved in pro-survival, mitogenic, apoptotic and growth regulation pathways in the progression from normal prostate epithelium to invasive prostate cancer. Using multiplexed reverse phase protein arrays coupled with LCM, the states of signaling changes during disease progression from prostate cancer study sets were analyzed. Focused analysis of phospho-specific endpoints revealed changes in cellular signaling events through disease progression and between patients. We have used a new protein array technology to study specific molecular pathways believed to be important in cell survival and progression from normal epithelium to invasive carcinoma directly from human tissue specimens. With the advent of molecular targeted therapeutics, the identification, characterization and monitoring of the signaling events within actual human biopsies will be critical for patient-tailored therapy. [source] Beta-catenin status in paediatric medulloblastomas: correlation of immunohistochemical expression with mutational status, genetic profiles, and clinical characteristics,THE JOURNAL OF PATHOLOGY, Issue 1 2009Sarah Fattet Abstract Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/,-catenin pathway occurs in 10-15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for ,-catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of ,-catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all ,-catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/,-catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear ,-catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/,-catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow-up of 75.7 months (range 27.5,121.2 months) from diagnosis. All three patients with focal nuclear staining of ,-catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1 -mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de-escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Vestibular Schwannoma Quantitative Polymerase Chain Reaction Expression of Estrogen and Progesterone Receptors,THE LARYNGOSCOPE, Issue 8 2008Andrew K. Patel MD Abstract Objectives/Hypothesis: Determine the role of estrogen receptor (ER) and progesterone receptor (PR) expression in sporadic and neurofibromatosis 2 (NF2)-related vestibular schwannomas (VS). Growth and proliferation signaling in human VS tumorigenesis may play a key role in molecular therapeutic targeting. VS carry mutations of the NF2 gene encoding the tumor suppressor, merlin, which interacts with ErbB2 in Schwann cells, implicating ErbB receptors in VS tumorigenesis. ErbB receptor family members are overexpressed or constitutively activated in many human tumors, and are effective therapeutic targets in some human cancers. VS occur more frequently in women and are larger, more vascular, and demonstrate increased growth rates during pregnancy. ER and PR may play a role in ErbB pathway activation and VS progression. Study Design: Quantitative real-time polymerase chain reaction (qRT-PCR) for ER and PR messenger RNA was performed using greater auricular and vestibular nerve controls (n = 8), sporadic VS (n = 23), and NF2-related VS (n = 16) tissues. Methods: The qRT-PCR data were normalized with standardization to a single constitutively expressed control gene, human cyclophylin. Results: Reverse transcription of messenger RNA from control and tumor specimens followed by RT Q-PCR demonstrated differences in ER and PR gene expression between sporadic and NF2-related VS. Conclusions: ER and PR expression in VS might have implications for development of a VS-specific drug delivery system using antihormone and ErbB pathway small molecule inhibitors, due to crosstalk between these receptors. These signals may be critical for re-establishing ErbB-mediated cell density dependent growth inhibition. [source] Restoration of PTEN expression alters the sensitivity of prostate cancer cells to EGFR inhibitors,THE PROSTATE, Issue 9 2008Z. Wu Abstract Introduction Prostate cancer (CaP) progression from an androgen-dependent to an androgen-independent state is associated with overexpression of EGFR family members or activation of their downstream signaling pathways, such as PI3K-Akt and MAPK. Although there are data implicating PI3K-Akt or MAPK pathway activation with resistance to EGFR inhibitors in CaP, the potential cross-talk between these pathways in response to EGFR or MAPK inhibitors remains to be examined. Methods Cross-talk between PTEN and MAPK signaling and its effects on CaP cell sensitivity to EGFR or MAPK inhibitors were examined in a PTEN-null C4-2 CaP cell, pTetOn PTEN C4-2, where PTEN expression was restored conditionally. Results Expression of PTEN in C4-2 cells exposed to EGF or serum was associated with increased phospho-ERK levels compared to cells without PTEN expression. Similar hypersensitivity of MAPK signaling was observed when cells were treated with a PI3K inhibitor LY294002. This enhanced sensitivity of MAPK signaling in PTEN-expressing cells was associated with a growth stimulatory effect in response to EGF. Furthermore, EGFR inhibitors gefitinib and lapatinib abrogated hypersensitivity of MAPK signaling and cooperated with PTEN expression to inhibit cell growth in both monolayer and anchorage-independent conditions. Similar cooperative growth inhibition was observed when cells were treated with the MEK inhibitor, CI1040, in combination with PTEN expression suggesting that inhibition of MAPK signaling could mediate the cooperation of EGFR inhibitors with PTEN expression. Conclusions Our results suggest that signaling cross-talk between the PI3K-Akt and MAPK pathways occurs in CaP cells, highlighting the potential benefit of targeting both the PI3K-Akt and MAPK pathways in CaP treatment. Prostate 68:935,944, 2008. © 2008 Wiley-Liss, Inc. [source] Heparin-binding epidermal growth factor-like growth factor functionally antagonizes interstitial cystitis antiproliferative factor via mitogen-activated protein kinase pathway activationBJU INTERNATIONAL, Issue 4 2009Jayoung Kim OBJECTIVE To delineate the mechanism underlying the potential functional relationship between interstitial cystitis antiproliferative factor (APF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF), as APF has previously been shown to decrease the proliferation rate of normal bladder epithelial cells and the amount of HB-EGF produced by these cells. MATERIALS AND METHODS APF-responsive T24 transitional carcinoma bladder cells were treated with high-pressure liquid chromatography-purified native APF with or without HB-EGF to determine the involvement of signalling pathways and proliferation by Western blot analysis, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (Erk)/MAPK assays, and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS Cyclic stretch induced the secretion of HB-EGF from T24 cells overexpressing the HB-EGF precursor, resulting in enhanced proliferation. T24 cells treated with APF had increased p38MAPK activity and suppressed cell growth, events that were both reversed by treatment with a p38MAPK-selective inhibitor. Activation of Erk/MAPK by HB-EGF was inhibited by APF, and APF did not stimulate p38MAPK in the presence of soluble HB-EGF or when cells overexpressed constitutively secreted HB-EGF. Lastly, APF inhibitory effects on cell growth were attenuated by HB-EGF. CONCLUSIONS These results indicate that HB-EGF and APF are functionally antagonistic and signal through parallel MAPK signalling pathways in bladder cells. [source] Prevalence and clinical correlates of JAK2 mutations in Down syndrome acute lymphoblastic leukaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2009Amos Gaikwad Summary Recurrent, prognostically significant chromosomal abnormalities occur in approximately 75% of paediatric acute lymphoblastic leukaemia (ALL), but only infrequently in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in the gene Janus kinase 2 (JAK2) were reported in 18% of DS ALL. Here we report identification and clinical correlates of JAK2 mutations in an independent cohort. JAK2 activating mutations occurred in 10/53 DS ALL cases (18·9%). Mutations were overrepresented in males (P < 0·03), occurred once in association with high hyperdiploidy and were not significantly correlated with age, initial white blood count, or event-free survival. Our results confirm the significance of JAK,STAT pathway activation in DS ALL. [source] Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts,BRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 7 2010J. P. Burke Background: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. Methods: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and/or transforming growth factor (TGF) ,1. Nuclear factor ,B (NF,B) pathway activation was assessed by inhibitory ,B, (I,B,) degradation and NF,B promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-,1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase,polymerase chain reaction and western blot. The NF,B pathway was inhibited by I,B, transfection. Results: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced I,B, degradation, enhanced NF,B promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-,1) significantly increased CTGF production relative to treatment with TGF-,1 alone. LPS reduced whereas TGF-,1 increased smad-7 expression. Transfection with an I,B, plasmid enhanced basal smad-7 expression. Conclusion: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NF,B and inducing collagen contraction. LPS acts in concert with TGF-,1 to induce CTGF. LPS reduces the expression of the TGF-,1 inhibitor, smad-7. Copyright © 2010 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] Wnt signaling stabilizes the DIXDC1 protein through decreased ubiquitin-dependent degradationCANCER SCIENCE, Issue 3 2010Lei Wang (Cancer Sci 2010; 101: 700,706) Wnt signaling plays key roles in development, cell growth, differentiation, polarity formation, neural development, and carcinogenesis. DIX Domain Containing 1 (DIXDC1), a novel component of the Wnt pathway, was recently cloned. DIXDC1 is the human homolog of Ccd1, a positive regulator of the Wnt signaling pathway during zebrafish neural patterning. Little has been known about DIXDC1 gene expression regulation. In the present study, we showed that the DIXDC1 protein was induced upon Wnt-3a stimulation, whereas the DIXDC1 mRNA level was not significantly increased after Wnt-3a treatment. Positive DIXDC1 staining was detected in colon cancer cells and was colocalized with ,-catenin staining. However, the DIXDC1 mRNA expression decreased in human colon cancer cells compared to the matched normal colon epithelial cells. Our further investigation showed that the DIXDC1 protein was degraded through the proteasome pathway, and the activation of canonical Wnt signaling decreased the ubiquitin-dependent degradation of both the ectopic and endogenous DIXDC1 protein. In order to explore the possible mechanism of the ubiquitination of DIXDC1, we found that the phosphorylation of DIXDC1 was inhibited by Wnt-3a. Collectively, these results indicate that canonical Wnt/,-catenin pathway activation might upregulate DIXDC1 through a post-translational mechanism by inhibiting the ubiquitin-mediated degradation of the DIXDC1 protein. [source] Hereditary diffuse gastric cancer: A manifestation of lost cell polarityCANCER SCIENCE, Issue 7 2009Bostjan Humar Hereditary diffuse gastric cancer is a cancer syndrome caused by germline mutations in the gene for the cell adhesion protein E-cadherin (CDH1). E-cadherin plays a central role in the maintenance of cell polarity and its loss during tumorigenesis is associated with poorly differentiated cancers and a poor prognosis. Hereditary diffuse gastric cancer is dominated by diffuse-type gastric adenocarcinoma, often with signet ring cell morphology. Large numbers of stage T1a signet ring cell carcinomas exist in the stomachs of CDH1 mutation carriers from a young age, and these foci sometimes show enrichment to the transition zone between the body and antrum. Generally these signet ring cell carcinomas are hypoproliferative, lack Wnt pathway activation, and are relatively indolent. However, a small proportion of the T1a foci contain cells that are poorly differentiated, display mesenchymal features, and express activated c-Src and its downstream targets. These same features are observed in more advanced stages of hereditary diffuse gastric cancer progression, suggesting that an epithelial,mesenchymal transition is required for tumor invasion beyond the muscularis mucosae. Hereditary diffuse gastric cancer initiation requires somatic down-regulation of the second CDH1 allele, which in most cases is caused by DNA promoter hypermethylation. Subsequent to CDH1 down-regulation, lost polarity in gastric stem or progenitor cells would be predicted to interfere with mitotic spindle orientation and the segregation of cell fate determinants. We predict that this disruption of cell division results in daughter cells being deposited in the lamina propria where their population expands and partially differentiates, resulting in the formation of foci of signet ring cells. (Cancer Sci 2009; 100: 1151,1157) [source] Design of a complement mannose-binding lectin pathway-specific activation system applicable at low serum dilutionsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2006M. Harboe Summary Recently we showed that alternative pathway (AP) amplification was responsible for more than 80% of specific classical pathway-induced terminal pathway activation under physiological conditions. The present study aimed to design a system for specific lectin pathway (LP) activation applicable at low serum dilutions with a fully functional AP. Comparison between activation of normal human serum (NHS), a mannose-binding lectin (MBL) homozygous D/D -deficient serum, and sera deficient in C1q and C2, all diluted 1 : 2, was essential to document optimal conditions for LP specificity. Mannan on the solid phase of enzyme-linked immunosorbent assay (ELISA) plates was used for activation, showing 0·5 µg mannan/well to give optimal conditions because at this concentration a good signal was preserved for C4 and TCC deposition in NHS, whereas the C3 deposition observed in C2-deficient serum at higher mannan concentrations reached nadir at 0·5 µg/well, indicating a lack of direct AP activation under these conditions. Pooled NHS and C1q-deficient serum gave the same degree of C4 and terminal complement complex (TCC) deposition, whereas deposition of these products was not obtained with MBL-deficient serum. Reconstitution with purified MBL, however, restored the depositions. A blocking anti-MBL monoclonal antibody (mAb) completely abolished the complement deposition, in contrast to a non-inhibiting anti-MBL mAb. Activation of C2-deficient serum induced C4 deposition similar to NHS, but negligible deposition of C3 and TCC, confirming the lack of direct activation of AP. Thus, this assay is unique in being LP-specific at low serum dilution and thus particularly suitable to study LP activation mechanisms and the role of AP amplification under physiological conditions. [source] | |||||||||||||||||||||||||||||||